Researchers develop new way to visualize single-cell protein secretion with amazing accuracy

We recently witnessed the stunning images of distant galaxies revealed by the James Webb Telescope, which were previously only visible as blurry spots. Researchers from Washington University in St. Louis have developed a new way to visualize proteins secreted by cells with amazing accuracy, making it the James Webb version of visualizing single-cell protein secretion.

The researchers, led by Srikanth Singamaneni, Lilyan & E. Professor of Mechanical Engineering and Materials Science, published an Aug. 5 article in the journal Cell Reports Methods. The highly sensitive test is able to see and measure the proteins secreted by a single cell in about 30 minutes.

In collaboration with researchers at the University of Washington School of Medicine and other universities, they have found that the FluoroDOT assay is versatile, low-cost, adaptable to any laboratory environment, and has the potential to provide a more comprehensive look at these proteins than current, widely used assays. Biomedical researchers search for these secreted proteins for information about cell-to-cell communication, cell signaling, activation and inflammation, among other actions, but current methods are limited in sensitivity and can take up to 24 hours to process.

What makes the FluoroDOT assay different from current assays is that it uses Fluoroplasmonic, a plasmon-enhanced nanomarker developed in the Singamaneni laboratory that is 16,000 times brighter than conventional fluorescence labels and has a signal-to-noise ratio about 30 times higher.

Plasmonic fluorophores consist of metallic nanoparticles that act as an antenna to draw light and enhance fluorescence emission from molecular fluorophores, making them ultra-bright nanoparticles.”

Srikanth Singamaneni, Lilyan & E. Lisle Hughes Professor of Mechanical Engineering and Materials Science, McKelvey School of Engineering

This ultra-bright emission of plasmonic fluorescence allows the user to see extremely small amounts of secreted protein, which they are unable to perform in current assays, and to quantify high-resolution signals digitally using the number of particles, or dot pattern, for each group, or spot, using a custom algorithm. In addition, they do not require special equipment. Singamaneni and his collaborators first published their work with plasmonic-fluor in Nature Biomedical Engineering in 2020.

The patent-pending Fluor-plasmonic technology has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC.

“Using a simple fluorescence microscope, we can simultaneously image a cell with the spatial distribution of the proteins secreted around it,” said Seth, who worked in Singamaneni’s lab and is now a lead scientist in cellular applications for Auragent Bioscience. “We have seen interesting patterns of secretion from different types of cells. This assay also allows simultaneous visualization of two types of proteins from individual cells. And when multiple cells are exposed to the same stimuli, we can distinguish cells that secrete two proteins at the same time from those that secrete only one or Not excreted at all.”

To validate the technique, the team used proteins secreted from human and mouse cells, including immune cells infected with Mycobacterium tuberculosis.

One of the collaborators and co-authors, Jennifer A. Phillips, MD, PhD, Theodore and Bertha Bryan Professor in the Departments of Medicine and Molecular Microbiology and co-director of the Division of Infectious Diseases at the College of Medicine, used the FluoroDOT assay in her laboratory.

“When M. tuberculosis infects immune cells, those cells respond by secreting important immune proteins called cytokines,” Phillips said. “But not all cells respond to infection in the same way. The FluoroDOT test allowed us to see how individual cells in a population respond to infection – to see which cells secrete and in what direction. This was not possible with the old technology.”

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Journal reference:

Seth, A.; et al. (2022) High-resolution imaging of protein secretion at the single cell level using the plasmon-enhanced FluoroDOT assay. Methods for cell reports. doi.org/10.1016/j.crmeth.2022.100267.

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